Method of producing a sustained-release preparation

ABSTRACT

A method of producing sustained-release microcapsules which comprises dispersing a physiologically active polypeptide into a solution of a biodegradable polymer and zinc oxide in an organic solvent, followed by removing the organic solvent; which provides a sustained-release preparation showing a high entrapment ratio of the physiologically active polypeptide and its constant high blood concentration levels over a long period of time.

TECHNICAL FIELD

The present invention relates to a method of producing asustained-release preparation comprising a physiologically activepolypeptide.

BACKGROUND ART

It is known that physiologically active polypeptides or theirderivatives exhibit a variety of pharmacological activities in vivo.Some of these have been produced on a large scale by utilizingEscherichia coli, yeasts, animal cells or host animals such as goat andhamsters using recently developed genetic engineering and celltechnology, and put to medicinal use. However, these physiologicallyactive polypeptides must be frequently administered because of thegenerally short biological half-life. The repeated injections takes asignificant physical burden on patients.

For instance, growth hormone (hereafter sometimes referred to as GH), arepresentative hormone which is originally produced and secreted in theanterior portion of the pituitary gland, is a physiologically activepolypeptide having widely diverse physiological activities such aspromotion of growth in the body, metabolism of glucose and lipids,anabolism of protein, and cell proliferation and differentiation. And GHis produced on a large scale by utilizing Escherichia coli using geneticrecombination technology, and put to medicinal use clinically andworldwidely. However, GH must be frequently administered in order tomaintain an effective blood concentration because of the shortbiological half-life. Especially, in the case of pituitary dwarfism, adaily subcutaneous administration to infants or young patients over along period of time ranging from a few months to at least 10 years isconducted practically.

To overcome this problem, various attempts have been made to develop asustained-release preparation comprising a physiologically activepolypeptide.

JP-A 3055/1996 (EP-A 633020) discloses a method of producing asustained-release preparation which comprises permitting a water-solublepolypeptide to permeate into a biodegradable matrix comprising abiodegradable polymer and a metal salt of a fatty acid in an aqueoussolution, and a sustained-release microcapsules (hereafter sometimesreferred to as MC) prepared by this method.

JP-A 217691/1996 (WO 96/07399) discloses production of a water-insolubleor slightly water-soluble polyvalent metal salt by using a water-solublepeptide type of physiologically active substance and an aqueous solutionof zinc chloride etc., and a method of producing a sustained-releasepreparation containing this salt and a biodegradable polymer.

WO 94/12158 discloses addition of a polymer erosion rate modulatingagent such as zinc hydroxide in an amount of 0.1 to 30% (w/w) relativeto the polymer to a polymer solution, as a method of producing asustained-release preparation comprising human GH and biodegradablepolymer. This publication further discloses a method of producing MC asporous particles by spraying a solution of human GH and a polymer in anorganic solvent into liquid nitrogen with biological activity retained.

WO 92/17200 and Nature Medicine, Vol. 2, p. 795 (1996) disclose a methodof producing a sustained-release preparation by using a zinc salt ofhuman GH.

WO 95/29664 discloses a method of producing MC which comprises the stepsof dispersing a metal salt such as zinc carbonate in a solid state in apolymer solution, adding a physiologically active substance such ashormones, and dispersing the physiologically active substance and ametal cation component separately through a biodegradable polymer.

Although, as described above, various attempts have been made to producea sustained-release preparation with a physiological activity of aphysiologically active polypeptide retained, a clinically satisfyingpreparation has not been obtained yet since some physiologically activepolypeptides have problems such as a low entrapment ratio of thephysiologically active polypeptide in the preparation, an excess releaseat an initial stage after administration, an unattained constant releaseover a long period of time, and an unretained satisfying bloodconcentration over a long period of time. Further, production methods,in many cases, are not suitable for industrialization which premises alarge-scale production in the present situation.

DISCLOSURE OF INVENTION

Through intensive investigation to resolve the above problems, thepresent inventors found that co-presence of lactic acid/glycolic acidcopolymer (hereafter sometimes referred to as PLGA) used as a MC baseand zinc oxide in an organic solvent unexpectedly provides dissolutionof zinc oxide which itself is insoluble in an organic solvent, andyields PLGA-zinc oxide complex efficiently in high contents, and that adirect dispersion of a physiologically active polypeptide in a solutionof the PLGA-zinc oxide complex in an organic solvent and subsequentmolding yields a sustained-release preparation having excellentproperties such as an enhanced entrapment ratio of the physiologicallyactive polypeptide, a reduced initial burst after administration, and anexcellent sustained-release. Further, the present inventors found thatthis production method has reduced a number of steps and is a quitesuitable method for industrialization. After further investigations, thepresent inventors developed the present invention.

Namely, the present invention relates to

(1) a method of producing a sustained-release preparation whichcomprises dispersing a physiologically active polypeptide into asolution of a biodegradable polymer and zinc oxide in an organicsolvent, followed by removing the organic solvent;

(2) the method according to the above (1), wherein the physiologicallyactive polypeptide is growth hormone;

(3) the method according to the above (1), wherein the biodegradablepolymer is lactic acid/glycolic acid copolymer;

(4) the method according to the above (3), wherein a molecularcomposition ratio of lactic acid/glycolic acid in the lacticacid/glycolic acid copolymer is about 85/15 to about 50/50;

(5) the method according to the above (3), wherein the weight-averagemolecular weight of the lactic acid/glycolic acid copolymer is about8,000 to about 20,000;

(6) the method according to the above (1), wherein the content of zincrelative to the biodegradable polymer in the organic solvent solution isabout 0.001 to about 2% by weight;

(7) the method according to the above (1), wherein the mean particlediameter of the sustained-release preparation is about 0.1 to about 300μm;

(8) the method according to the above (1), wherein the sustained-releasepreparation is for injection;

(9) the method according to the above (1), wherein an o/w emulsioncomprising a dispersion prepared by dispersing growth hormone into asolution of lactic acid/glycolic acid copolymer and zinc oxide in anorganic solvent as an oil phase is subjected to in-water drying;

(10) the method according to the above (1), wherein the preparation is amicrocapsule;

(11) a solution of lactic acid/glycolic acid copolymer and zinc oxide inan organic solvent;

(12) a lactic acid/glycolic acid copolymer-zinc oxide complex which issoluble in an organic solvent and which is obtained by co-presence oflactic acid/glycolic acid copolymer and zinc oxide in an organicsolvent;

(13) a dispersion which is prepared by dispersing a physiologicallyactive polypeptide into a solution of lactic acid/glycolic acidcopolymer and zinc oxide in an organic solvent;

(14) the dispersion according to the above (13), wherein thephysiologically active polypeptide is growth hormone; and

(15) the sustained-release preparation which is produced by the methodaccording to the above (1).

Preferable examples of the biodegradable polymers used in the presentinvention include polymers synthesized from one or moreα-hydroxycarboxylic acids (e.g., glycolic acid, lactic acid),hydroxydicarboxylic acids (e.g., malic acid), hydroxytricarboxylic acids(e.g., citric acid) etc. by catalyst-free dehydration condensationpolymerization and having a free terminal carboxyl group, mixturesthereof, poly-α-cyanoacrylates, polyamino acids (e.g.,poly-γ-benzyl-L-glutamic acid) and maleic anhydride copolymers (e.g.,styrene-maleic acid copolymers).

Polymerization may be of the random, block or graft type. When theabove-mentioned α-hydroxycarboxylic acids, hydroxydicarboxylic acids andhydroxytricarboxylic acids have an optical active center in theirmolecular structures, they may be of the D-, L- or DL-configuration.

Among these polymers, a biodegradable polymer having a free terminalcarboxyl group such as polymers synthesized from α-hydroxycarboxylicacids (e.g., glycolic acid, lactic acid) (e.g., lactic acid/glycolicacid copolymer) and poly-α-cyanoacrylates are preferred. Thebiodegradable polymer is more preferably a polymer synthesized fromα-hydroxycarboxylic acids, especially preferably lactic acid/glycolicacid copolymer.

When the biodegradable polymer used is a lactic acid/glycolic acidcopolymer or homopolymer, its composition ratio (mol %) is preferablyabout 100/0 to about 40/60, more preferably about 85/15 to about 50/50.

In the present specification, lactic acid/glycolic acid copolymer aswell as homopolymers such as polylactic acid and polyglycolic acid issometimes simply referred to as lactic acid/glycolic acid polymer.

The weight-average molecular weight of the above-described lacticacid/glycolic acid polymer is preferably about 3,000 to about 25,000,more preferably about 5,000 to about 20,000.

The degree of dispersion (weight-average molecular weight/number-averagemolecular weight) of the lactic acid/glycolic acid polymer is preferablyabout 1.2 to about 4.0, more preferably about 1.5 to about 3.5.

Regarding weight-average molecular weight and degree of dispersion, thepresent specification holds that the former is in terms of polystyreneas determined by gel permeation chromatography (GPC) using 9polystyrenes as reference substances with weight-average molecularweights of 120,000, 52,000, 22,000, 9,200, 5,050, 2,950, 1,050, 580 and162, respectively, and that the latter is calculated therefrom. Theabove determination was carried out using a GPC column KF804Lx2(produced by Showa Denko, Japan) and an RI monitor L-3300 (produced byHitachi, Ltd., Japan) with chloroform as a mobile phase.

A biodegradable polymer having a free terminal carboxyl group is abiodegradable polymer wherein the number-average molecular weight basedon GPC measurement and the number-average molecular weight based onterminal group quantitation almost agree with each other.

The number-average molecular weight based on terminal group quantitationis calculated as follows:

About 1 to 3 g of the biodegradable polymer is dissolved in a mixedsolvent of acetone (25 ml) and methanol (5 ml), and the solution isquickly titrated with a 0.05 N alcoholic solution of potassium hydroxidewhile stirring at room temperature (20° C.) with phenolphthalein as anindicator to determine the carboxyl group content; the number-averagemolecular weight based on terminal group quantitation is calculated fromthe following equation:

Number-average molecular weight based on terminal groupquantitation=20000×A/B

A: Weight mass (g) of biodegradable polymer

B: Amount (ml) of the 0.05 N alcoholic solution of potassium hydroxideadded until titration end point is reached

While the number-average molecular weight based on terminal groupquantitation is an absolute value, that based on GPC measurement is arelative value that varies depending on various analytical conditions(e.g., kind of mobile phase, kind of column, reference substance, slicewidth chosen, baseline chosen etc.); it is therefore difficult to havean absolute numerical representation of these two values. However, thedescription that the number-average molecular weight based on GPCmeasurement and that based on terminal group quantitation almost agreemeans, for instance, that the number-average molecular weight based onterminal group quantitation falls within the range from about 0.5 toabout 2 times, preferably from about 0.7 to about 1.5 times, of thenumber-average molecular weight based on GPC measurement in a polymerwhich is synthesized from one or more α-hydroxycarboxylic acids.

For example, in the case of a polymer having a free terminal carboxylgroup and which is synthesized from one or more α-hydroxycarboxylicacids by catalyst-free dehydration condensation polymerization, thenumber-average molecular weight based on GPC measurement and thenumber-average molecular weight based on terminal group quantitationalmost agree with each other. On the other hand, in the case of apolymer having substantially no free terminal carboxyl groups and whichis synthesized from a cyclic dimer by ring-opening polymerization usinga catalyst, the number-average molecular weight based on terminal groupquantitation is significantly (about 2 times or more) higher than thatbased on GPC measurement. This difference makes it possible to clearlydifferentiate a polymer having a free terminal carboxyl group from apolymer having no free terminal carboxyl group.

A lactic acid/glycolic acid polymer having a free terminal carboxylgroup can be produced by a per se known process such as that describedin JP-A 28521/1986 (e.g., process by catalyst-free dehydrationcondensation polymerization reaction or dehydration condensationpolymerization reaction in the presence of an inorganic solid acidcatalyst).

The decomposition/elimination rate of a lactic acid/glycolic acidpolymer varies widely, depending on composition rate or weight-averagemolecular weight. Drug release duration can be extended by lowering theglycolic acid ratio or increasing the molecular weight, sincedecomposition/elimination is usually delayed as the glycolic acid ratiodecreases. Conversely, drug release duration can be shortened byincreasing the glycolic acid ratio or decreasing the molecular weight.To obtain a long-term (e.g., 1-4 months) sustained-release preparation,it is preferable to use a lactic acid/glycolic acid polymer whosecomposition ratio and weight-average molecular weight are within theabove-described ranges.

Therefore, in the present invention, composition of a biodegradablepolymer used is preferably selected according to the desired kinds of aphysiologically active polypeptide and the desired duration. In aspecific example, for instance, when GH is used as a physiologicallyactive polypeptide, lactic acid/glycolic acid copolymer is preferablyused. In the lactic acid/glycolic acid copolymer, lactic acid/glycolicacid composition ratio (mol %) is preferably about 85/15 to about 50/50,more preferably about 75/25 to about 50/50. The weight-average molecularweight of the lactic acid/glycolic acid copolymer is preferably about8,000 to about 20,000, more preferably about 10,000 to about 20,000.Further, the degree of dispersion (weight-average molecularweight/number-average molecular weight) of the lactic acid/glycolic acidcopolymer is about 1.2 to about 4.0, more preferably about 1.5 to about3.5.

The lactic acid/glycolic acid copolymer used can be produced by theknown methods such as those described in the above publication and thelike. The copolymer is preferably one that is produced by catalyst-freedehydration polymerization. It is preferable that the lacticacid/glycolic acid copolymer (PLGA) wherein the number-average molecularweight based on terminal group quantitation and the number-averagemolecular weight based on GPC measurement almost agree with each otheris used.

Further, two kinds of lactic acid/glycolic acid copolymers differing incomposition ratio and weight-average molecular weight may be used in anadmixture of given ratio. The typical example is a mixture of lacticacid/glycolic acid copolymer wherein the composition ratio of lacticacid/glycolic acid (mol %) is about 75/25 and the weight-averagemolecular weight is about 10,000 and lactic acid/glycolic acid copolymerwherein the composition ratio of lactic acid/glycolic acid (mol %) isabout 50/50 and the weight-average molecular weight is about 12,000. Thepreferred weight ratio of the mixture is about 25/75 to about 75/25.

In the present invention, zinc oxide used for preparation of PLGA-zincoxide complex is a little water-soluble zinc compound, and is itselfinsoluble or slightly soluble also in an organic solvent such asdichloromethane. The co-presence of zinc oxide and PLGA in an organicsolvent such as dichloromethane quite unexpectedly provides formation ofPLGA-zinc oxide complex efficiently and the subsequent dissolution inthe organic solvent. These operations are accomplished simply byaddition of PLGA and zinc oxide in the organic solvent, and do not needa separation process of PLGA-zinc oxide complex. To the thus obtainedsolution of PLGA-zinc oxide complex in an organic solvent, aphysiologically active polypeptide is directly added to produce MCcontaining the physiologically active polypeptide easily. Further, thethus obtained MC maintains the physiologically active polypeptidebiologically stable, and provides a sustained-release preparation whichshows a reduced initial release and an excellent sustained release.

The physiologically active polypeptide used in the present inventionincludes a physiologically active polypeptide having molecular weight ofpreferably about 1,000 to about 50,000, more preferably about 5,000 toabout 40,000.

The representative activity of the physiologically active polypeptide ishormonal activity. The physiologically active polypeptide may be naturalproducts, synthetic products, semi-synthetic products, and theirderivatives and analogues. The mode of action of the physiologicallyactive polypeptide may be agonistic or antagonistic.

The physiologically active polypeptide for use in the present inventionincludes peptide hormones, cytokines, peptide neurotransmitters,hematopoietic factors, various growth factors, enzymes, polypeptideantibiotics and analgesic peptides.

Examples of the peptide hormones include insulin, somatostatin,somatostatin derivative (Sandostatin; see U.S. Pat. Nos. 4,087,390,4,093,574, 4,100,117 and 4,253,998), growth hormones (GH), sodiumdiuretic peptides, gastrin, prolactin, adrenocorticotropic hormone(ACTH), ACTH derivatives (e.g., ebiratide), melanocyte-stimulatinghormone (MSH), thyrotropin-releasing hormone (TRH) and salts andderivatives thereof (see JP-A 121273/1975 and 116465/1977),thyroid-stimulating hormone (TSH), luteinizing hormone (LH),follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG),thymosin, motilin, vasopressin, vasopressin derivative [desmopressin,see Folia Endocrinologica Japonica, Vol. 54, No. 5, pp. 676-691 (1978)],oxytocin, calcitonin, parathyroid hormone (PTH), glucagon, secretin,pancreozymin, cholecystokinin, angiotensin, and human placentallactogen. The peptide hormones are preferably insulin and growthhormones.

The cytokines include lymphokines and monokines. The lymphokines includeinterferons (alpha, beta and gamma) and interleukins (IL-2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12). The monokines include interleukin-1 (IL-1), andtumor necrosis factor (TNF). The preferred cytokine is a lymphokine and,more preferred interferon. The particularly preferred cytokine isinterferon-α.

The peptide neurotransmitters include substance P, serotonin and GABA.

The hematopoietic factors include erythropoietin (EPO), colonystimulating factors (G-CSF, GM-CSF, M-CSF), thrombopoietin (TPO),platelet-derived growth factor, and megakaryocyte potentiator.

The various growth factors include basic and acidic fibroblast growthfactors (FGF) and their families (e.g., EGF, TGF-α, TGF-β, PDGF, acidicFGF, basic FGF, FGF-9), nerve growth factor (NGF) and its family (e.g.,BDNF, NT-3, NT-4, CNTF, GDNF), insulin-like growth factors (e.g. IGF-1,IGF-2, etc.) and bone morphogenetic protein (BMP) and its family.

The enzymes include superoxide dismutase (SOD), urokinase, tissueplasminogen activator (TPA), asparaginase and kallikrein.

The polypeptide antibiotics include polymixin B, colistin, gramicidinand bacitracin.

The analgesic peptides include enkephalin, enkephalin derivatives (seeU.S. Pat. No. 4,277,394 and EP-A 31567), endorphin, and kyotorphin.

Further, the physiologically active polypeptides include thymopoietin,dynorphin, bombesin, caerulein, thymostimulin, thymic humoral factor(THF), blood thymic factor (FTS) and derivatives thereof (see U.S. Pat.No. 4,229,438), other thymic factors [Igaku no Ayumi, Vol. 125, No. 10,pp. 835-843 (1983)], neurotensin, bradykinin, andendothelin-antagonistic peptides (see EP-A 436189, 457195 and 496452,and JP-A 94692/1991 and 130299/1991).

The particularly preferred physiologically active polypeptides includegrowth hormone and insulin.

In the present invention, when the physiologically active polypeptidecontains a metal, the content of the metal is preferably not greaterthan 0.1%, more preferably not greater than 0.01%, and most preferablynot greater than 0.001%. Thus, substantially metal-free physiologicallyactive polypeptides are most suited for the present invention.Crystalline insulin, for instance, usually contains small amounts ofheavy metals such as zinc, nickel, cobalt and cadmium. Insulincontaining 0.4% (w/w) zinc exists as a stable hexamer and appears to berelatively inert in the interaction with the biodegradable polymer metalsalt.

If necessary, the metals occurring in the physiologically activepolypeptide may be previously removed. As the method of removing themetals, known methods are employed. For example, dialyzing an acidicaqueous hydrochloric acid solution of insulin against water or anaqueous solution of ammonium acetate and lyophilizing the dialysateprovide amorphous insulin with minimal metal content.

Growth hormone may be derived from any species, and it is preferably onederived from human beings. Further, although natural products extractedfrom the pituitary gland can be used for the present invention, geneticrecombinant type GH (JP-B 12996/1994, 48987/1994) is preferred. Therecombinant type human GH having the same structure with a natural typewithout methionine at the N-terminal group is more preferred. Such GHmay be in the form of a metal salt, and one containing substantially nometal is also used.

The organic solvent used in the present invention preferably has aboiling point not exceeding 120° C. Such organic solvent includeshalogenated hydrocarbons (e.g., dichloromethane, chloroform, carbontetrachloride, etc.), alcohols (e.g. ethanol, methanol, 1,4-butanediol,1,5-pentanediol, etc.), ethyl acetate, acetonitrile, and so on. Thesesolvents can also be used as a mixture in a given ratio. The preferredorganic solvent used singly includes, for instance, dichloromethane andacetonitrile.

The preferred organic solvent used as a mixture includes, for instance,combination of halogenated hydrocarbons (e.g., dichloromethane,chloroform) and alcohols (e.g., ethanol, methanol, 1,4-butanediol,1,5-pentanediol) or acetonitrile. Especially, combination ofdichloromethane and acetonitrile is used widely. The mixing ratio(volume ratio) of halogenated hydrocarbons relative to alcohols oracetonitrile ranges from about 40:1 to about 1:1, preferably from about20:1 to about 1:1. Especially, single use of halogenated hydrocarbonssuch as dichloromethane is preferred.

In the present invention, zinc oxide used for production of asustained-release preparation is preferably in a fine powder state.Although the reaction time is expected to become shorter as the diameteris smaller, problems of handling arise at the same time since fryabilityincreases. The particle diameter of zinc oxide is usually about 0.001 μmto about 10 μm, preferably about 0.005 μm to about 1 μm, more preferablyabout 0.01 μm to about 0.1 μm.

In a solution of a biodegradable polymer and zinc oxide in an organicsolvent, the content of zinc (Zn) by weight ratio relative to thebiodegradable polymer is preferably about 0.001 to about 2% (w/w), morepreferably about 0.01 to about 2% (w/w), most preferably about 0.1 toabout 2% (w/w). The content of zinc in the solution of the biodegradablepolymer and zinc oxide in the organic solvent is determined by commonlyknown analytical methods such as the atomic absorption analysis.

In the present specification, the sustained-release preparation is notlimited as long as it is in the form of fine particles comprising aphysiologically active polypeptide and a microcapsule base (i.e., abiodegradable polymer-zinc oxide complex). Examples of the fineparticles include microcapsules containing one drug core in eachparticles, multiple-core microcapsules containing a large number of drugcores in each particles, microspheres in which a drug in a molecularform is dissolved or dispersed in the microcapsule base.

The sustained-release preparation in the present invention is producedby dispersing a physiologically active polypeptide into a solution of abiodegradable polymer and zinc oxide in an organic solvent, and thenremoving the organic solvent. In the present specification, a productwhich is produced from a biodegradable polymer and zinc oxide, andformed in a clear solution wherein the biodegradable polymer and zincoxide are dissolved in the organic solvent is referred to as “abiodegradable polymer-zinc oxide complex”.

The biodegradable polymer-zinc oxide complex may be a compound resultingfrom intermolecular interaction such as normal salts, complex salts,double salts and organic metal compounds, or a composition.

The biodegradable polymer-zinc oxide complex has properties such asdissolution in an organic solvent and provision of an excellentsustainability to the sustained-release preparation as a final product.Further, the complex in which a biodegradable polymer is PLGA isreferred to as “a PLGA-zinc oxide complex”.

In the present specification, “dispersion” means homogeneous dispersionof a physiologically active polypeptide in an organic solvent. Bothsolution and suspension of the physiologically active polypeptide in theorganic solvent are included in the dispersion of the present invention.

In the method of the present invention, methods of removing the organicsolvent include, for instance, (a) in-water drying method (o/w method),(b) phase separation method (coacervation method), (c) spray-dryingmethod, and modifications thereof.

Hereafter, production method when microcapsules are produced as asutained-release preparation is described.

In the method of the present invention, a biodegradable polymer and zincoxide are first allowed to exist together in an organic solvent toprepare a solution of a biodegradable polymer-zinc oxide complex in theorganic solvent. Although the concentration of the biodegradable polymerin the solution varies depending on the molecular weight and the kindsof the organic solvent, it is, for instance, about 0.1 to about 80%(w/w), preferably about 1 to about 70% (w/w), more preferably about 2 toabout 60% (w/w). The amount of zinc oxide added varies depending on thekinds of the organic solvent, and is, for instance, about 0.001 to about5% (w/w), preferably about 0.01 to about 2.5% (w/w), more preferablyabout 0.1 to about 2.5% (w/w), based on the amount of the biodegradablepolymer.

Regarding the order of addition of the biodegradable polymer and zincoxide to the organic solvent, zinc oxide both in a powder state or in adispersed state in the organic solvent can be added to a solution of thebiodegradable polymer in the organic solvent, conversely, a solution ofthe biodegradable polymer in the organic solvent can be added to adispersion of zinc oxide in the organic solvent. Further, the organicsolvent can be added after the biodegradable polymer and zinc oxide bothin a powder state are admixed.

The conditions to produce a solution of a biodegradable polymer-zincoxide complex such as PLGA-zinc oxide complex from a biodegradablepolymer and zinc oxide can be changed according to the kinds of thebiodegradable polymer used, the particle diameter of zinc oxide, thekinds of the organic solvent, these compositions. For instance, whenPLGA is employed as a polymer, PLGA-zinc oxide complex can be obtainedby the reaction usually at about 0 to about 30° C., preferably about 2to about 25° C., for about 1 to about 168 hours, preferably about 12 toabout 96 hours, more preferably about 24 to about 72 hours. However, thereaction time is not limited to the above ranges and can be determinedusing as an index liquid state observations by the naked eye. Theproduciton of PLGA-zinc oxide complex in the present invention can beconfirmed by the naked eye since zinc oxide which is in a dispersedstate at the time of addition dissolves in the organic solvent to give aclear solution.

Although this reaction proceeds simply by the co-presence of PLGA andzinc oxide in the organic solvent, the reaction carried out understirring or shaking by means of suitable stirring or shaking means isadvantageous to reduction of the reaction time. Further, the reactioncarried out under ultrasonication is equally preferred. As the reactiontemperature becomes higher, the reaction time becomes shorter. While thehigher reaction temperature is at the same time accompanied with thefaster degradation of PLGA.

The thus obtained biodegradable polymer-zinc oxide complex is applied tothe next process preferably as a solution in an organic solvent, ifnecessary as a solid after removal of the organic solvent.

Then, a physiologically active polypeptide preferably in a powder stateis added for dissolution or dispersion in an amount of about 0.1 toabout 50% (w/w), preferably about 1 to about 20% (w/w), more preferablyabout 3 to about 15% (w/w), to the solution of the biodegradable polymerand zinc oxide in the organic solvent to produce a dispersion of thebiodegradable polymer, zinc oxide and the physiologically activepolypeptide in an organic solvent (hereafter simply referred to as aphysiologically active polypeptide dispersion).

If the physiologically active polypeptide has properties such as nodissolution in the solution of the biodegradable polymer and zinc oxidein the organic solvent, turbidity by addition in a powder state, andhardness to be dispersed, it is preferred that the physiologicallyactive polypeptide is previously dispersed in the organic solvent. Tothe organic solvent solution, stabilizers for the physiologically activepolypeptide (e.g., serum albumin, gelatin, protamine sulfate) can beadded.

For the purpose of homogeneous dispersion of the physiologically activepolypeptide in the organic solvent, addition of external physical energyis preferred. Such methods include, for instance, ultrasonication,turbine-type stirrer and homogenizer. In this case, the particle size ofthe physiologically active polypeptide in the organic solvent is about0.01 to about 200 μm, preferably about 0.05 to about 100 μm, morepreferably about 0.1 to about 50 μm. The concentration of thephysiologically active polypeptide in the organic solvent is about 1 toabout 50%, preferably about 2 to about 20%. Such treatment providesuniformity in the particle size of the physiologically activepolypeptide in the organic solvent, and homogeneous dispersion in asolution of the biodegradable polymer and zinc oxide in the organicsolvent.

Further, the physiologically active polypeptide can be previouslydispersed in the organic solvent independently of the biodegradablepolymer-zinc oxide complex. In this case, the composition of the organicsolvent used and that of the organic solvent used for dissolution of thebiodegradable polymer and zinc oxide may be the same or different fromeach other. For instance, it is possible that the biodegradablepolymer-zinc oxide complex is dissolved in dichloromethane, thephysiologically active polypeptide is dispersed in acetonitrile, andthen both are admixed. In this case, the ratio (volume ratio) of thephysiologically active polypeptide to the biodegradable polymer-zincoxide complex is, for instance, about 1:1,000 to about 1:1, preferablyabout 1:200 to about 1:5, especially preferably about 1:100 to about1:5.

(a) In-water drying method (o/w method)

The dispersion of the physiologically active polypeptide prepared in theabove manner is further added to an aqueous phase to form an o/wemulsion. Then the solvent in the oil phase is volatilized to producemicrocapsules. In this case, an emulsifier may be added to the externalaqueous phase. The emulsifier may be any substance capable of providingfor a stable o/w emulsion. Examples of such emulsifiers include anionicsurfactants, nonionic surfactants, polyoxyethylene-castor oilderivatives, polyvinylpyrrolidone, polyvinyl alcohol,carboxymethylcellulose, lecithin, gelatin, hyaluronic acid and so on.The preferred emulsifier is polyvinyl alcohol. These emulsifiers may beused singly or in combination of two or more. The concentration of theemulsifier in the external aqueous phase ranges from about 0.001 toabout 20% (w/w), preferably from about 0.01 to about 10% (w/w),especially preferably from about 0.05 to about 5% (w/w).

The thus obtained microcapsules are recovered by centrifugation orfiltration, washed with distilled water to remove the emulsifier etc.adhering to the surface of microcapsules, redispersed in distilledwater, and lyophilized.

Then, if necessary, water and the organic solvent in the microcapsulesare further removed by heating. The heating may be conducted underreduced pressure. Regarding the heating conditions, heating and dryingare conducted at a temperature not lower than a glass transitiontemperature of the biodegradable polymer and not so high as to causeaggregation of each microcapsule particle. The heating and drying areconducted preferably at a temperature ranging from the glass transitiontemperature of the biodegradable polymer to a temperature which is about30° C. higher than the glass transition temperature. The glasstransition temperature is defined as the intermediate glass transitionpoint obtained using a differential scanning calorimeter when thetemperature is increased at a rate of 10 to 20° C. per minute.

(b) Phase separation method (Coacervation method)

When MC is produced by the present method, a coacervating agent isgradually added to the above described dispersion of the physiologicallyactive polypeptide under stirring to precipitate and solidify MC. Theamount of the coacervating agent used is about 0.01 to about 1,000 timesby volume, preferably about 0.05 to about 500 times by volume,especially preferably about 0.1 to about 200 times by volume. Anycoacervating agent can be used, as long as it is a polymeric, mineraloil or vegetable oil compound miscible with the organic solvent fordissolution of a biodegradable polymer and it does not dissolve thebiodegradable polymer used. Specifically, examples of such coacervatingagents include silicone oil, sesame oil, soybean oil, corn oil,cottonseed oil, coconut oil, linseed oil, mineral oil, n-hexane andn-heptane. Two or more of these may be used in combination.

The thus obtained MC are recovered by filtration, washed repeatedly withheptane etc. to remove the coacervating agent. Further, washing isconducted in the same manner as in the above (a), followed bylyophilization.

In the production of MC by the in-water drying method or coacervationmethod, an antiaggregation agent may be added in the process of washingMC for preventing aggregation of particles. Examples of theantiaggregation agent include, for instance, water-solublepolysaccharides such as mannitol, lactose, glucose, starches (e.g., cornstarch), hyaluronic acid and its alakaline metal salt; amino acids suchas glycine and alanine; proteins such as fibrin and collagen; andinorganic salts such as sodium chloride and sodium hydrogen phosphate,and so on.

(c) Spray-drying method

When MC is produced by the present method, the dispersion of thephysiologically active polypeptide is sprayed via a nozzle into thedrying chamber of a spray drier to volatilize the organic solvent in thefine droplets in a very short time to yield MC. Examples of the nozzleinclude, for instance, a two-fluid nozzle type, a pressure nozzle typeand a rotary disc type. It is also advantageous, if necessary, to sprayan aqueous solution of the above-described antiaggregation agent viaanother nozzle in order to prevent aggregation of each MC particle.

The thus obtained MC is washed in the same manner as in the above (a),if necessary followed by heating (if necessary under reduced pressure)to remove water and the organic solvent.

In the present invention, the entrapment ratio of the physiologicallyactive polypeptide such as GH into MC is preferably at least 50% whenPLGA-zinc oxide complex is employed as a microcapsule base.

The contents of the physiologically active polypeptide in thesustained-release preparation of the present invention are, forinstance, about 0.1 to about 30% (w/w), preferably about 0.2 to about20% (w/w), more preferably about 0.5 to about 10% (w/w).

The sustained-release preparation of the present invention can beadministered, for instance, as fine particles such as microcapsules assuch, or in the form of various dosage forms of non-oral preparations(e.g., intramuscular, subcutaneous or visceral injections; indwellablepreparations; nasal, rectal or uterine transmucosal preparations etc.)or oral preparations (e.g., capsules such as hard capsules and softcapsules etc.; solid preparations such as granules and powders etc.;liquid preparations such as suspensions etc.) by using the fineparticles as a raw material.

The preparations of these dosage forms can be produced by known methodsin common use for pharmaceutical production.

The sustained-release preparation of the present invention is preferablyin the form of injections. To prepare injections using the fineparticles such as microcapsules obtained by the above methods, the fineparticles may be formulated with a dispersant (e.g., surfactants such asTween 80, HCO-60; polysaccharides such as carboxymethylcellulose, sodiumalginate, sodium hyaluronate; protamine sulfate; polyethylene glycol400, etc.), a preservative (e.g., methyl paraben, propyl paraben, etc.),an isotonizing agent (e.g., sodium chloride, mannitol, sorbitol,glucose, etc.), and a local anesthetic (e.g., xylocaine hydrochloride,chlorobutanol, etc.), to provide an aqueous suspension, or dispersedwith vegetable oil (e.g., sesame oil, corn oil, etc.), or a mixturethereof with a phospholipid (e.g., lecithin) or medium-chain fatty acidtriglycerides (e.g., Miglyol 812) to provide an oily suspension.

The sustained-release preparation is especially preferably in the formof fine particles. The particle diameter of the sustained-releasepreparation for an injectable suspension may be selected from the rangesatisfying the requirements for the degree of dispersion and the needlepassability for the injection. For instance, the particle diameter iswithin the range of about 0.1 to about 300 μm, preferably about 1 toabout 150 μm, more preferably about 2 to about 100 μm, as the meanparticle diameter.

Methods of preparing the above fine particles as a sterile preparationinclude, but are not limited to, the method in which the entireproduction process is sterile, the method in which the gamma rays areused as the sterilant, and the method in which an antiseptic is addedduring the manufacturing process.

The sustained-release preparation can be safely used in mammals (e.g.,humans, bovine, swine, dogs, cats, mice, rats, rabbits, etc.) with lowtoxicity.

Indication of the sustained-release preparation varies depending on thephysiologically active polypeptide used. The sustained-releasepreparation is useful to prevent or treat diabetes when insulin is usedas the physiologically active polypeptide; viral hepatitis (e.g., type Chepatitis, HBe antigen-positive active hepatitis) and cancer (e.g.,renal carcinoma, multiple myeloma, etc.) when interferon-a is used;anemia (e.g., anemia during dialysis of kidney) when erythropoietin isused; neutropenia (e.g., in cancer therapy) and infections when G-CSF isused; cancer (e.g., hemangioendothelioma) when IL-2 is used; fracture,wound (e.g., bedsore), periodontitis and gastrointestinal ulcer when FGFis used; thrombocytopenia when FGF-9 is used; senile dementia andneuropathy when NGF is used; thrombosis when TPA is used; and cancerwhen tumor necrosis factor is used.

Further, the sustained-release preparation containing GH is applied toTurner's syndrome, chronic renal diseases, achondroplasia, and adulthypopituitarism as well as pituitary dwarfism, based on growth hormoneaction of GH. Further, GH is reported to be applied to diseases such asDown syndrome, Silver syndrome, hypochondroplasia and juvenile chronicarthritis to provide excellent therapeutic effects.

Although varing depending on the kinds and contents of thephysiologically active polypeptide, duration of the release, targetdisease, subject animal species and other factors, the dose of thesustained-release preparation may be set at any level, as long as theeffective concentration of the physiologically active polypeptide in thebody is maintained.

For instance, when the sustained-release preparation is one designed forone week release, the dose of the physiologically active polypeptide canbe chosen from the range of preferably about 0.0001 to about 10 mg/kgbody weight, more preferably about 0.0005 to about 1 mg/kg body weight,per an adult. The preferred administration frequency of thesustained-release preparation can be suitably chosen from once a week,once every two weeks and etc. depending on the kinds and contents of thephysiologically active polypeptide, the dosage form, duration of therelease, target disease, subject animal species and other factors.

When the physiologically active polypeptide as an active ingredient inthe sustained-release preparation is, for instance, insulin, the doseper administration to an diabetic adult is chosen from the range ofusually about 0.001 to about 1 mg/kg body weight, preferably about 0.01to about 0.2 mg/kg body weight, as an effective ingredient. And thepreferred administration frequency is once a week.

When the physiologically active polypeptide as an active ingredient inthe sustained-release preparation is GH, the dose may be set at anylevel, as long as the effective concentration of GH in the body ismaintained, although varing depending on the kinds and contents of GH,duration of the release, target disease, subject animal species andother factors. Regarding the treatment of the above described diseases,when the sustained-release preparation is one designed for two weekrelease, the dose of GH can be chosen from the range of about 0.01 toabout 5 mg/kg body weight, more preferably about 0.05 to about 1 mg/kgbody weight, per a child or an adult for safe administration. Thepreferred administration frequency can be suitably chosen from once aweek, once every two weeks, once a month and etc., depending on thecontents of GH, the dosage form, duration of the release, targetdisease, subject animal species and other factors.

The sustained-release preparation is preferably stored at ordinarytemperature or in cold place. More preferably, the sustained-releasepreparation is stored in cold place. The “ordinary temperature” and the“cold place” are defined in the pharmacopoeia of Japan. Namely, the“ordinary temperature” means 15 to 25° C., and the “cold place” means atemperature not exceeding 15° C.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is hereafter described in more detail by means ofthe following Working Examples and Experimental Examples, which are notto be construed to limit the scope of the present invention.

WORKING EXAMPLE 1

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 10,000) and 6.6 mg ofzinc oxide were added to 1.7 ml of dichloromethane, which was stirred(60 rpm) at 25° C. for 3 days to yield a clear solution of lacticacid/glycolic acid copolymer-zinc oxide complex in an organic solvent.To this solution was added 53.0 mg of a freeze-dried powder of humangrowth hormone, and they were admixed by means of a voltex mixer and asmall-size homogenizer. Then, an ultrasonication was carried out toyield a solution of human growth hormone and lactic acid/glycolic acidcopolymer-zinc oxide complex in an organic solvent. This organic solventsolution was poured into 400 ml of a 0.1% (w/v) polyvinyl alcohol (PVA)aqueous solution previously adjusted at 18° C., followed byemulsification with a turbine-type homomixer to yield an o/w emulsion.This o/w emulsion was stirred at room temperature to volatilizedichloromethane. The obtained microcapsules were collected bycentrifugation (about 1,500 rpm). The obtained precipitate was washedtwice with 400 ml of distilled water, which was freeze-dried to yield521 mg of powdery microcapsules containing human growth hormone.

WORKING EXAMPLE 2

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 10,000) and 13.1 mgof zinc oxide were dissolved in 2.3 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 53.3 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 536 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 3

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 10,000) and 21.9 mgof zinc oxide were dissolved in 2.8 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 53.8 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 589 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 4

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 12,000) and 5.1 mg ofzinc oxide were dissolved in 1.9 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 52.9 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 506 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 5

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 12,000) and 10.2 mgof zinc oxide were dissolved in 2.5 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 53.2 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 568 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 6

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=65/35(mol %), weight-average molecular weight 12,000) and 17.0 mgof zinc oxide were dissolved in 3.0 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 53.5 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 561 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 7

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 15,000) and 4.5 mg ofzinc oxide were dissolved in 2.0 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 52.9 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 540 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 8

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 15,000) and 8.9 mg ofzinc oxide were dissolved in 2.6 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 53.1 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 559 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 9

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 15,000) and 14.9 mgof zinc oxide were dissolved in 3.1 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 53.4 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 464 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 10

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 20,000) and 4.0 mg ofzinc oxide were dissolved in 2.5 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 52.8 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 595 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 11

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 20,000) and 7.9 mg ofzinc oxide were dissolved in 3.6 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 53.1 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 478 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 12

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 20,000) and 13.2 mgof zinc oxide were dissolved in 5.2 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 53.3 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 534 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 13

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=75/25(mol %), weight-average molecular weight 10,500) and 6.6 mg ofzinc oxide were dissolved in 3.0 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 53.0 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 521 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 14

1 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=85/15(mol %), weight-average molecular weight 12,000) and 5.8 mg ofzinc oxide were dissolved in 2.0 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 53.0 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 503 mg of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 15

1.89 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 10,000) and 10 mg ofzinc oxide were dissolved in 3.4 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 100 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 1.41 g of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 16

1.89 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 12,000) and 10 mg ofzinc oxide were dissolved in 3.5 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 100 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 1.41 g of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 17

1.89 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 14,000) and 10 mg ofzinc oxide were dissolved in 4.0 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 100 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 1.40 g of powdery microcapsules containinghuman growth hormone.

WORKING EXAMPLE 18

1.89 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 16,000) and 10 mg ofzinc oxide were dissolved in 4.2 ml of dichloromethane to yield asolution of lactic acid/glycolic acid copolymer-zinc oxide complex in anorganic solvent. To this solution was added 100 mg of a freeze-driedpowder of human growth hormone, which was treated in the same manner asin Working Example 1 to yield 1.34 g of powdery microcapsules containinghuman growth hormone.

COMPARATIVE EXAMPLE 1

Lactic acid/glycolic acid copolymer (lactic acid/glycolic acid=50/50(mol%), weight-average molecular weight 15,000) was dissolved indichloromethane (950 mg/ml) to yield a solution of lactic acid/glycolicacid copolymer in an organic solvent. 1 ml of this solution and 1 ml ofa solution of a freeze-dried powder of human growth hormone indichloromethane (50 mg/ml) were mixed, which was treated in the samemanner as in Working Example 1 to yield 490 mg of powdery microcapsulescontaining human growth hormone.

COMPARATIVE EXAMPLE 2

1.90 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 10,000) was dissolvedin 2.6 ml of dichloromethane to yield a solution of lactic acid/glycolicacid copolymer in an organic solvent. To this solution was added 100 mgof a freeze-dried powder of human growth hormone, which was treated inthe same manner as in Working Example 1 to yield 1.28 g of powderymicrocapsules containing human growth hormone.

COMPARATIVE EXAMPLE 3

1.90 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 12,000) was dissolvedin 2.8 ml of dichloromethane to yield a solution of lactic acid/glycolicacid copolymer in an organic solvent. To this solution was added 100 mgof a freeze-dried powder of human growth hormone, which was treated inthe same manner as in Working Example 1 to yield 1.18 g of powderymicrocapsules containing human growth hormone.

COMPARATIVE EXAMPLE 4

1.90 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 14,000) was dissolvedin 3.0 ml of dichloromethane to yield a solution of lactic acid/glycolicacid copolymer in an organic solvent. To this solution was added 100 mgof a freeze-dried powder of human growth hormone, which was treated inthe same manner as in Working Example 1 to yield 0.89 g of powderymicrocapsules containing human growth hormone.

COMPARATIVE EXAMPLE 5

1.90 g of lactic acid/glycolic acid copolymer (lactic acid/glycolicacid=50/50(mol %), weight-average molecular weight 16,000) was dissolvedin 3.2 ml of dichloromethane to yield a solution of lactic acid/glycolicacid copolymer in an organic solvent. To this solution was added 100 mgof a freeze-dried powder of human growth hormone, which was treated inthe same manner as in Working Example 1 to yield 1.26 g of powderymicrocapsules containing human growth hormone.

EXPERIMENTAL EXAMPLE 1

308 mg of microcapsules containing human growth hormone and PLGA-zincoxide complex obtained in Working Example 7; 351 mg of microcapsulescontaining human growth hormone and PLGA-zinc oxide complex obtained inWorking Example 8; 327 mg of microcapsules containing human growthhormone and PLGA-zinc oxide complex obtained in Working Example 9; and229 mg of microcapsules containing human growth hormone and PLGAobtained in Comparative Example 1; were dispersed in respectively 2.25ml of dispersion medium (composition of the dispersion medium: mannitol(5%), carboxymethylcellulose (0.5%) and Tween 20 (0.1%) were dissolvedin distilled water, followed by adjustment of pH to 6.0 with aceticacid) (the same shall apply hereinafter), 2.25 ml of dispersion medium,1.75 ml of dispersion medium, and 1.75 ml of dispersion medium.

0.5 ml of the thus obtained dispersion (containing 3 mg of human GH) wassubcutaneously administered at the back of rats under anesthesia withether. Blood was collected via tail vein with the passage of time andblood serum was separated. The human GH concentration in the obtainedblood serum was determined by means of a radioimmunoassay (Ab Beads HGH,produced by Eiken Kagaku, Japan). The obtained results are shown inTable 1.

TABLE 1 Sustained-release preparation Blood Concentration (ng/ml)containing growth 1st 2nd 4th 6th 7th 9th 11th hormone day day day dayday day day Working Ex. 7 16.7 8.6 8.4 14.8 26.6 13.5 2.8 Working Ex. 817.9 10.6 14.1 22.5 27.8 15.9 3.5 Working Ex. 9 14.0 8.5 15.8 33.9 28.217.8 7.7 Comparative Ex. 1 4.3 1.7 2.1 3.1 5.0 8.0 2.7

The human GH concentration in groups in which microcapsules containinghuman growth hormone and PLGA-zinc oxide complex obtained in WorkingExamples 7, 8 and 9 were administered showed significantly high valuescompared with that in a group in which microcapsules containing humangrowth hormone and PLGA obtained in Comparative Example 1 wereadministered, and further showed a long-term sustained-release.According to the method of the present invention, a sustained-releasepreparation with an excellent release profile can be produced.

EXPERIMENTAL EXAMPLE 2

550 mg, 556 mg, 576 mg and 573 mg of microcapsules containing humangrowth hormone and PLGA-zinc oxide complex respectively obtained inWorking Examples 15, 16, 17 and 18 were dispersed in 3.38 ml of thedispersion medium described in Experimental Example 1. While 548 mg, 548mg, 567 mg and 560 mg of microcapsules containing human growth hormoneand PLGA obtained in Comparative Examples 2, 3, 4 and 5 were dispersedin 3.38 ml of the same dispersion medium.

0.75 ml of the thus obtained dispersion (containing 6 mg of human GH)was subcutaneously administered at the back of rats under anesthesiawith ether. Blood was collected via tail vein with the passage of timeand blood serum was separated. The human GH concentration in theobtained blood serum was determined by means of the radioimmunoassaydescribed in Experimental Example 1. The obtained results are shown inTables 2 to 5.

TABLE 2 Sustained-release preparation Blood Concentration (ng/ml)containing 4th 7th 9th 11th growth hormone day day day day Working Ex.15 24.8 24.5 15.2 6.3 Comparative Ex. 2 13.8  8.1  5.1 3.9

TABLE 3 Sustained-release preparation Blood Concentration (ng/ml)containing 4th 7th 9th 11th growth hormone day day day day Working Ex.16 24.6 25.3 16.3 8.7 Comparative Ex. 3  9.2 10.1  9.6 6.8

TABLE 4 Sustained-release preparation Blood Concentration (ng/ml)containing 4th 7th 9th 11th growth hormone day day day day Working Ex.17 12.5 35.4 29.5 9.7 Comparative Ex. 4  7.6 14.8  7.7 6.2

TABLE 5 Sustained-release preparation Blood Concentration (ng/ml)containing 4th 7th 9th 11th growth hormone day day day day Working Ex.18 11.8 31.8 35.5 11.3 Comparative Ex. 5  4.9 13.8 18.5  6.7

The human GH concentration in groups in which microcapsules containinghuman growth hormone and PLGA-zinc oxide complex obtained in WorkingExamples 15, 16, 17 and 18 were administered showed significantly highvalues compared with that in groups in which microcapsules containinghuman growth hormone and PLGA obtained in Comparative Examples 2, 3, 4and 5 were administered.

Industrial Applicability

According to the present invention, a sustained-release preparationwhich shows a high entrapment ratio of a physiologically activepolypeptide such as growth hormone and its constant high bloodconcentration levels over a long period of time can be provided.

What is claimed is:
 1. A method of producing a sustained-releasepreparation which comprises dispersing a physiologically activepolypeptide into a solution of a lactic acid/glycolic acid copolymer,wherein a molecular composition ratio of lactic acid/glycolic acid inthe acid/glycolic acid copolymer is about 85/15 to about 50/50 and zincoxide in an organic solvent, followed by removing the organic solvent.2. The method according to claim 1, wherein the physiologically activepolypeptide is growth hormone.
 3. The method according to claim 1,wherein the weight-average molecular weight of the lactic acid/glycolicacid copolymer is about 8,000 to about 20,000.
 4. The method accordingto claim 1, wherein the content of zinc relative to the lacticacid/glycolic acid copolymer, wherein a molecular composition ratio oflactic acid/glycolic acid in the lactic acid/glycolic acid copolymer isabout 85/15 to about 50/50 biodegradable polymer in the organic solventsolution is about 0.001 to about 2% by weight.
 5. The method accordingto claim 1, wherein the mean particle diameter of the sustained-releasepreparation is about 0.1 to about 300 μm.
 6. The method according toclaim 1, wherein the sustained-release preparation is for injection. 7.The method according to claim 1, wherein an o/w emulsion comprising adispersion prepared by dispersing growth hormone into a solution oflactic acid/glycolic acid copolymer and zinc oxide in an organic solventas an oil phase is subjected to in-water drying.
 8. The method accordingto claim 1, wherein the preparation is a microcapsule.
 9. A dispersionwhich is prepared by dispersing a physiologically active polypeptideinto a solution of lactic acid/glycolic acid copolymer and zinc oxide inan organic solvent.
 10. The dispersion according to claim 9, wherein thephysiologically active polypeptide is growth hormone.
 11. Thesustained-release preparation which is produced by the method accordingto claim 1.